Inverse Expression Relationship Research Articles

Transcriptome-wide analysis uncovers regulatory elements of the antennal transcriptome repertoire of bumblebee at different life stages.

Bumblebees are crucial pollinators, providing essential ecosystem services and global food production. The success of pollination services relies on the interaction between sensory organs and the environment. The antenna functions as a versatile multi-sensory organ, pivotal in mediating chemosensory/olfactory information, and governs adaptive responses to environmental changes. Despite an increasing number of RNA-sequencing studies on insect antenna, comprehensive antennal transcriptome studies at the different life stages were not elucidated systematically. Here, we quantified the expression profile and dynamics of coding/microRNA genes of larval head and antennal tissues from early- and late-stage pupa to the adult of Bombus terrestris as suitable model organism among pollinators. We further performed Pearson correlation analyses on the gene expression profiles of the antennal transcriptome from larval head tissue to adult stages, exploring both positive and negative expression trends. The positively correlated coding genes were primarily enriched in sensory perception of chemical stimuli, ion transport, transmembrane transport processes and olfactory receptor activity. Negatively correlated genes were mainly enriched in organic substance biosynthesis and regulatory mechanisms underlying larval body patterning and the formation of juvenile antennal structures. As post-transcriptional regulators, miR-1000-5p, miR-13b-3p, miR-263-5p and miR-252-5p showed positive correlations, whereas miR-315-5p, miR-92b-3p, miR-137-3p, miR-11-3p and miR-10-3p exhibited negative correlations in antennal tissue. Notably, based on the inverse expression relationship, positively and negatively correlated microRNA (miRNA)-mRNA target pairs revealed that differentially expressed miRNAs predictively targeted genes involved in antennal development, shaping antennal structures and regulating antenna-specific functions. Our data serve as a foundation for understanding stage-specific antennal transcriptomes and large-scale comparative analysis of transcriptomes in different insects.

Investigating miR-9 as a mediator in laryngeal cancer health disparities.

For several decades, Black patients have carried a higher burden of laryngeal cancer among all races. Even when accounting for sociodemographics, a disparity remains. Differentially expressed microRNAs have been linked to racially disparate clinical outcomes in breast and prostate cancers, yet an association in laryngeal cancer has not been addressed. In this study, we present our computational analysis of differentially expressed miRNAs in Black compared with White laryngeal cancer and further validate microRNA-9-5p (miR-9-5p) as a potential mediator of cancer phenotype and chemoresistance. Bioinformatic analysis of 111 (92 Whites, 19 Black) laryngeal squamous cell carcinoma (LSCC) specimens from the TCGA revealed miRNAs were significantly differentially expressed in Black compared with White LSCC. We focused on miR-9-5 p which had a significant 4-fold lower expression in Black compared with White LSCC (p<0.05). After transient transfection with either miR-9 mimic or inhibitor in cell lines derived from Black (UM-SCC-12) or White LSCC patients (UM-SCC-10A), cellular migration and cell proliferation was assessed. Alterations in cisplatin sensitivity was evaluated in transient transfected cells via IC50 analysis. qPCR was performed on transfected cells to evaluate miR-9 targets and chemoresistance predictors, ABCC1 and MAP1B. Northern blot analysis revealed mature miR-9-5p was inherently lower in cell line UM-SCC-12 compared with UM-SCC-10A. UM -SCC-12 had baseline increase in cellular migration (p < 0.01), proliferation (p < 0.0001) and chemosensitivity (p < 0.01) compared to UM-SCC-10A. Increasing miR-9 in UM-SCC-12 cells resulted in decreased cellular migration (p < 0.05), decreased proliferation (p < 0.0001) and increased sensitivity to cisplatin (p < 0.001). Reducing miR-9 in UM-SCC-10A cells resulted in increased cellular migration (p < 0.05), increased proliferation (p < 0.05) and decreased sensitivity to cisplatin (p < 0.01). A significant inverse relationship in ABCC1 and MAP1B gene expression was observed when miR-9 levels were transiently elevated or reduced in either UM-SCC-12 or UM-SCC-10A cell lines, respectively, suggesting modulation by miR-9. Collectively, these studies introduce differential miRNA expression in LSCC cancer health disparities and propose a role for low miR-9-5p as a mediator in LSCC tumorigenesis and chemoresistance.

Dysregulation of miRNA and its reversal with l-ascorbic acid during AGE-BSA induced ER stress in Mus musculus neuronal cells

Loss of function and progressive death of neurons, the pathological mechanism of neurodegenerative diseases (NDDs) can occur as a result of apoptotic unfolded protein response (UPR) signaling induced by endoplasmic reticulum (ER) stress resulting from misfolded protein accumulation in ER lumen. Non-enzymatic glycation of proteins have been implicated in the pathophysiology of NDDs and are known to cause cellular oxidative stress leading to ER stress mediated neuronal apoptosis. MicroRNAs (miRNAs), post transcriptional regulators of gene expression are identified as regulators of UPR and the altered brain miRNA profiles in NDDs suggest the involvement of miRNA dysregulation in neuronal death. l-ascorbic acid, a micro nutrient and an important neuroprotectant has been suggested to be a modulator of gene expression. However there are few reports on miRNAs targeting the genes involved in neuronal apoptotic UPR signaling as well as l-ascorbic acid mediated regulation of neuronal UPR pathway and miRNA expression. Here, we identified a statistically significant downregulation of 7 miRNAs such as mmu-miR-24, 27b, 124, 224, 290, 351 and 488 and inverse expression relationship with their UPR related targets such as Perk, Ire1α, Chop and Puma during advanced glycated BSA (AGE-BSA) induced ER stress in Mus musculus cortical and hippocampal neurons. We also observed that a pretreatment with l-ascorbic acid alleviated AGE-BSA induced neuronal ER stress as well as the downregulation of the 7 potential miRNAs.

A Comprehensive Repertoire of Transfer RNA-Derived Fragments and Their Regulatory Networks in Colorectal Cancer.

To provide systematic insight into the composition and expression of transfer RNA (tRNA) derivatives transcriptome in colorectal cancer (CRC). tRNA derivatives expression profiles in three pairs of CRC and adjacent normal colon tissues were performed by tRNA-derived small RNA fragments (tRFs) and tRNA halves (tiRNA) sequencing, and microarray data of transcriptomes from CRC and paired controls were retrieved from Gene Expression Omnibus database. The differentially expressed tRFs and tiRNAs and differentially expressed genes between CRC and paired normal samples were screened. The functional regulations between tRF and tiRNA and gene were identified. A total of 60 upregulated and 48 downregulated tRNA derivatives and 7373 upregulated and 12,138 downregulated messenger RNA (mRNA) were identified. The tRF and tiRNA-gene regulatory modules were constructed by analyzing computational tRF and tiRNA-target predictions and inverse expression relationships between tRF and tiRNAs and mRNA. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway annotation showed that the function of targets of tiRNA-Tyr-GTA was mainly enriched in negative regulation of epithelial cell apoptotic process and peroxisome proliferator activated-receptors (PPAR) signaling pathway. Cellular response to monoamine stimulus and inflammatory bowel disease was enriched in function of tiRNA-Val-CAC. Two functions, including negative regulation of c-Jun N-terminal kinase (JNK) cascade and choline metabolism in cancer, were enriched in tRF-Gln-CTG. The function of mesenchymal to epithelial transition was enriched in tRF-Leu-TAG. For the first time to our knowledge, our study provided a landscape of tRNA derivatives expression profiles in CRC. Further tRF and tiRNA-gene regulatory modules construction explored the potential functions related to these tRNA derivatives in the pathogenesis of CRC.

Oxidative Stress Down-Regulates MiR-20b-5p, MiR-106a-5p and E2F1 Expression to Suppress the G1/S Transition of the Cell Cycle in Multipotent Stromal Cells.

Oxidative stress has been linked to senescence and tumorigenesis via modulation of the cell cycle. Using a hydrogen peroxide (H2O2)-induced oxidative stress-induced premature senescence (OSIPS) model previously reported by our group, this study aimed to investigate the effects of oxidative stress on microRNA (miRNA) expression in relation to the G1-to-S-phase (G1/S) transition of the cell cycle and cell proliferation. On global miRNA analysis of the OSIPS cells, twelve significantly up- or down-regulated miRNAs were identified, the target genes of which are frequently associated with cancers. Four down-regulated miR-17 family miRNAs are predicted to target key pro- and anti-proliferative proteins of the p21/cyclin D-dependent kinase (CDK)/E2F1 pathway to modulate G1/S transition. Two miR-17 miRNAs, miR-20-5p and miR-106-5p, were confirmed to be rapidly and stably down-regulated under oxidative stress. While H2O2 treatment hampered G1/S transition and suppressed DNA synthesis, miR-20b-5p/miR-106a-5p over-expression rescued cells from growth arrest in promoting G1/S transition and DNA synthesis. Direct miR-20b-5p/miR-106a-5p regulation of p21, CCND1 and E2F1 was demonstrated by an inverse expression relationship in miRNA mimic-transfected cells. However, under oxidative stress, E2F1 expression was down-regulated, consistent with hampered G1/S transition and suppressed DNA synthesis and cell proliferation. To explain the observed E2F1 down-regulation under oxidative stress, a scheme is proposed which includes miR-20b-5p/miR-106a-5p-dependent regulation, miRNA-E2F1 autoregulatory feedback and E2F1 response to repair oxidative stress-induced DNA damages. The oxidative stress-modulated expression of miR-17 miRNAs and E2F1 may be used to develop strategies to retard or reverse MSC senescence in culture, or senescence in general.

Identification of Cross Talk between FoxM1 and RASSF1A as a Therapeutic Target of Colon Cancer.

Metastatic colorectal cancer (mCRC) is characterized by the expression of cellular oncogenes, the loss of tumor suppressor gene function. Therefore, identifying integrated signaling between onco-suppressor genes may facilitate the development of effective therapy for mCRC. To investigate these pathways we utilized cell lines and patient derived organoid models for analysis of gene/protein expression, gene silencing, overexpression, and immunohistochemical analyses. An inverse relationship in expression of oncogenic FoxM1 and tumor suppressor RASSF1A was observed in various stages of CRC. This inverse correlation was also observed in mCRC cells lines (T84, Colo 205) treated with Akt inhibitor. Inhibition of FoxM1 expression in mCRC cells as well as in our ex vivo model resulted in increased RASSF1A expression. Reduced levels of RASSF1A expression were found in normal cells (RWPE-1, HBEpc, MCF10A, EC) stimulated with exogenous VEGF165. Downregulation of FoxM1 also coincided with increased YAP phosphorylation, indicative of tumor suppression. Conversely, downregulation of RASSF1A coincided with FoxM1 overexpression. These studies have identified for the first time an integrated signaling pathway between FoxM1 and RASSF1A in mCRC progression, which may facilitate the development of novel therapeutic options for advanced colon cancer therapy.

Inverse and correlative relationships between TRIBBLES genes indicate non-redundant functions during normal and malignant hemopoiesis

TRIBBLES pseudokinases (TRIB1, TRIB2, and TRIB3) are important regulators of normal and malignant hemopoiesis. The relative abundance of each TRIBBLES family member may be important for distinct oncogenic or tumor suppressor functions. We map the expression profiles of TRIB1, TRIB2, and TRIB3 in human and murine hemopoietic stem, progenitor and mature cells, and in human leukemia datasets. Our data show that TRIB1-TRIB2 have an inverse expression relationship in normal hemopoiesis, whereas TRIB1-TRIB3 have a positive correlation. We reveal that TRIB3 expression is high in the dormant hemopoietic stem cell (HSC) population, implicating a novel role for TRIB3 in stem cell quiescence. These analyses support a non-redundant role for each TRIBBLES member during normal hemopoietic differentiation. We show that TRIB1-TRIB2 display a significant negative correlation in myelodysplastic syndrome and acute myeloid leukemia (AML) subtypes, but not in acute lymphoid leukemia. This inverse relationship is specific to certain subtypes of AML. A positive correlation exists in different leukemia subtypes between TRIB1-TRIB3. The TRIB1-TRIB2 and TRIB1-TRIB3 correlations are consistent with a correlative relationship with C/EBP transcription factor family members. Our results have implications for the development of strategies to therapeutically target these genes in different types of leukemia.

BRN2, a POUerful driver of melanoma phenotype switching and metastasis.

The POU domain family of transcription factors play a central role in embryogenesis and are highly expressed in neural crest cells and the developing brain. BRN2 is a class III POU domain protein that is a key mediator of neuroendocrine and melanocytic development and differentiation. While BRN2 is a central regulator in numerous developmental programs, it has also emerged as a major player in the biology of tumourigenesis. In melanoma, BRN2 has been implicated as one of the master regulators of the acquisition of invasive behaviour within the phenotype switching model of progression. As a mediator of melanoma cell phenotype switching, it coordinates the transition to a dedifferentiated, slow cycling and highly motile cell type. Its inverse expression relationship with MITF is believed to mediate tumour progression and metastasis within this model. Recent evidence has now outlined a potential epigenetic switching mechanism in melanoma cells driven by BRN2 expression that induces melanoma cell invasion. We summarize the role of BRN2 in tumour cell dissemination and metastasis in melanoma, while also examining it as a potential metastatic regulator in other tumour models.

Expression Profiling Analysis Reveals Key MicroRNA-mRNA Interactions in Early Retinal Degeneration in Retinitis Pigmentosa.

PurposeThe aim of this study was to identify differentially expressed microRNAs (miRNAs) that might play an important role in the etiology of retinal degeneration in a genetic mouse model of retinitis pigmentosa (rd10 mice) at initial stages of the disease.MethodsmiRNAs–mRNA interaction networks were generated for analysis of biological pathways involved in retinal degeneration.ResultsOf more than 1900 miRNAs analyzed, we selected 19 miRNAs on the basis of (1) a significant differential expression in rd10 retinas compared with control samples and (2) an inverse expression relationship with predicted mRNA targets involved in biological pathways relevant to retinal biology and/or degeneration. Seven of the selected miRNAs have been associated with retinal dystrophies, whereas, to our knowledge, nine have not been previously linked to any disease.ConclusionsThis study contributes to our understanding of the etiology and progression of retinal degeneration.

Identification of differentially expressed miRNAs and miRNA-targeted genes in bladder cancer.

BackgroundDifferentially expressed genes and their post-transcriptional regulator-microRNAs (miRNAs), are potential keys to pioneering cancer diagnosis and treatment. The aim of this study was to investigate how the miRNA-mRNA interactions might affect the tumorigenesis of bladder cancer (BC) and to identify specific miRNA and mRNA genetic markers in the two BC types: non-muscle invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC).ResultsWe identified 227 genes that interacted with 54 miRNAs in NMIBC, and 14 genes that interacted with 10 miRNAs in MIBC. Based on this data, we found extracellular matrix-related genes are highly enriched in NMIBC while genes related to core nuclear division are highly enriched in MIBC. Furthermore, using a transcriptional regulatory element database, we found indirect regulatory transcription factors (TFs) for enriched genes could regulate tumorigenesis with or without miRNAs.Materials and methodsTissue samples from 234 patients histologically diagnosed with BC and 83 individuals without BC were analyzed using microarray and next-generation sequencing technology, and we used different cut-offs to identify differentially expressed mRNAs and miRNAs in NMIBC and MIBC. The selected mRNAs and miRNAs were paired using validated target datasets and according to inverse expression relationships. MiRNA interacted genes were compared with the TF-regulating genes in BC. Meanwhile, pathway enrichment analysis was performed to identify the functions of selected miRNAs and genes.ConclusionsIdentification of differential gene expression in specific tumor types could facilitate development of cancer diagnosis and aid in the early detection of BC.

MiR-524-5p of the primate-specific C19MC miRNA cluster targets TP53IPN1- and EMT-associated genes to regulate cellular reprogramming

BackgroundIntroduction of the transcription factors Oct4, Sox2, Klf4, and c-Myc (OSKM) is able to ‘reprogram’ somatic cells to become induced pluripotent stem cells (iPSCs). Several microRNAs (miRNAs) are known to enhance reprogramming efficiency when co-expressed with the OSKM factors. The primate-specific chromosome 19 miRNA cluster (C19MC) is essential in primate reproduction, development, and differentiation. miR-524-5p, a C19MC member, is highly homologous to the reprogramming miR-520d-5p; we also reported that miR-524-5p was expressed in iPSCs but not mesenchymal stem cells (MSCs). This study aimed to elucidate possible contributions of miR-524-5p to the reprogramming process.MethodsA miR-524-5p precursor was introduced into human fibroblast HFF-1 in the presence of OSKM, and the relative number of embryonic stem cell (ESC)-like colonies that stained positively with alkaline phosphatase (AP) and Nanog were quantified to determine reprogramming efficiency. A miR-524-5p mimic was transfected to MSCs to investigate the effects of miR-524-5p on TP53INP1, ZEB2, and SMAD4 expression by real-time polymerase chain reaction (PCR) and Western blot. Direct gene targeting was confirmed by luciferase activity. A phylogenetic tree of TP53INP1 was constructed by the Clustal method. Contribution of miR-524-5p to cell proliferation and apoptosis was examined by cell counts, BrdU, MTT, and cell death assays, and pluripotency gene expression by real-time PCR.ResultsCo-expressing the miR-524 precursor with OSKM resulted in a two-fold significant increase in the number of AP- and Nanog-positive ESC-like colonies, indicating a role for miR-524-5p in reprogramming. The putative target, TP53INP1, showed an inverse expression relationship with miR-524-5p; direct TP53INP1 targeting was confirmed in luciferase assays. miR-524-5p-induced TP53INP1 downregulation enhanced cell proliferation, suppressed apoptosis, and upregulated the expression of pluripotency genes, all of which are critical early events of the reprogramming process. Interestingly, the TP53INP1 gene may have co-evolved late with the primate-specific miR-524-5p. miR-524-5p also promoted mesenchymal-to-epithelial transition (MET), a required initial event of reprogramming, by directly targeting the epithelial-to-mesenchymal transition (EMT)-related genes, ZEB2 and SMAD4.ConclusionsVia targeting TP53INP1, ZEB2, and SMAD4, miR-524-5p contributes to the early stage of inducing pluripotency by promoting cell proliferation, inhibiting apoptosis, upregulating expression of pluripotency genes, and enhancing MET. Other C19MC miRNAs may have similar reprogramming functions.

Integrated analysis of microRNA and gene expression profiles reveals a functional regulatory module associated with liver fibrosis

BackgroundLiver fibrosis, characterized with the excessive accumulation of extracellular matrix (ECM) proteins, represents the final common pathway of chronic liver inflammation. Ever-increasing evidence indicates microRNAs (miRNAs) dysregulation has important implications in the different stages of liver fibrosis. However, our knowledge of miRNA-gene regulation details pertaining to such disease remains unclear. MethodsThe publicly available Gene Expression Omnibus (GEO) datasets of patients suffered from cirrhosis were extracted for integrated analysis. Differentially expressed miRNAs (DEMs) and genes (DEGs) were identified using GEO2R web tool. Putative target gene prediction of DEMs was carried out using the intersection of five major algorithms: DIANA-microT, TargetScan, miRanda, PICTAR5 and miRWalk. Functional miRNA-gene regulatory network (FMGRN) was constructed based on the computational target predictions at the sequence level and the inverse expression relationships between DEMs and DEGs. DAVID web server was selected to perform KEGG pathway enrichment analysis. Functional miRNA-gene regulatory module was generated based on the biological interpretation. Internal connections among genes in liver fibrosis-related module were determined using String database. MiRNA-gene regulatory modules related to liver fibrosis were experimentally verified in recombinant human TGFβ1 stimulated and specific miRNA inhibitor treated LX-2 cells. ResultsWe totally identified 85 and 923 dysregulated miRNAs and genes in liver cirrhosis biopsy samples compared to their normal controls. All evident miRNA–gene pairs were identified and assembled into FMGRN which consisted of 990 regulations between 51 miRNAs and 275 genes, forming two big sub-networks that were defined as down-network and up-network, respectively. KEGG pathway enrichment analysis revealed that up-network was prominently involved in several KEGG pathways, in which “Focal adhesion”, “PI3K-Akt signaling pathway” and “ECM-receptor interaction” were remarked significant (adjusted p<0.001). Genes enriched in these pathways coupled with their regulatory miRNAs formed a functional miRNA-gene regulatory module that contains 7 miRNAs, 22 genes and 42 miRNA-gene connections. Gene interaction analysis based on String database revealed that 8 out of 22 genes were highly clustered. Finally, we experimentally confirmed a functional regulatory module containing 5 miRNAs (miR-130b-3p, miR-148a-3p, miR-345-5p, miR-378a-3p, and miR-422a) and 6 genes (COL6A1, COL6A2, COL6A3, PIK3R3, COL1A1, CCND2) associated with liver fibrosis. ConclusionsOur integrated analysis of miRNA and gene expression profiles highlighted a functional miRNA-gene regulatory module associated with liver fibrosis, which, to some extent, may provide important clues to better understand the underlying pathogenesis of liver fibrosis.

Patterns of Microrna in Plasma Cells: From Normal Differentiation to Multiple Myeloma

Background: MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression either via the degradation of target mRNAs or the inhibition of protein translation. miRNAs have been involved in fine-tuning critical cellular processes and play critical roles in cell differentiation and tumor development. Furthermore, there is an increasing recognition of the role of miRNAs in multiple myeloma, a plasma cell (PC) malignancy characterized by an accumulation of malignant PCs within the bone marrow. However, little is known about miRNA expression during human PCD as well as about the full extent to which individual miRNAs regulate fundamental processes during PCD. A complete delineation of miRNA and their target expression during normal PCD is essential to understand the role of miRNAs in plasma cell malignancies.Methods: We analyzed the expression profile of miRNAs and mRNAs during human plasma cell differentiation (PCD) to infer miRNA-target relationships, as well as in multiple myeloma tumor plasma cells. We developed a method and an R package, that uses miRNA and mRNA expression profiles across PCD cell subpopulations to infer candidate miRNA-target interactions that could be active and functional in PCD. We inferred miRNA-target relationships from sequence-based prediction, experimentally validated target interactions curated from the literature, published data from miRNA perturbation experiments and inverse expression relationships between miRNAs and their target mRNAs.Results: Our results reveal 63 miRNAs with significant temporal changes in their expression during normal PCD. We derived a high-confidence network of 295 target relationships comprising 47 miRNAs and 147 targets. These relationships include new examples of miRNAs that are likely to coordinately regulate multiple members of critical pathways associated with PCD. Notably, we identify new miRNAs that coordinately regulate important pathways in PCD, including members of miR-30 and miR-29 families, miR-106b and miR-16, which regulates IRF4/PRDM1 axis, active DNA methylation pathway, TGF-b signaling pathway, autophagy, ZBTB4/EZH2 axis and cell cycle progression. Furthermore, our work demonstrates that 28 PCD stage-specific miRNAs are aberrantly overexpressed in multiple myeloma cells (MMCs) compared to their normal counterpart and/or are associated with high risk myeloma, suggesting that MMCs frequently acquired expression changes in miRNAs already undergoing dynamic expression modulation during normal PCD. Finally, expression of some targets of these PCD - MM miRNAs is correlated with clinical outcome of uniformly treated MM patients.Conclusions: Altogether, our results demonstrate that miRNAs may be important in controlling PCD and malignant plasma cell biology. DisclosuresNo relevant conflicts of interest to declare.

Immunohistochemical angiogenic biomarkers in hepatocellular carcinoma and cirrhosis: correlation with pathological features

OBJECTIVE:To investigate immunohistochemical markers of angiogenesis and their association with pathological prognostic features in hepatocellular carcinoma and cirrhotic liver.METHODS:Vascular endothelial growth factor, CD105, and cyclooxygenase-2 were immunohistochemically detected in 52 hepatocellular carcinoma tissue samples and 48 cirrhotic liver tissue samples. Semiquantitative measurements of vascular endothelial growth factor and cyclooxygenase-2 were evaluated considering the degree and intensity of immunostaining based on a 7-point final scoring scale. CD105 microvascular density (MVD-CD105) was measured using automated analysis. Morphological aspects evaluated in the hepatocellular carcinoma samples included size (≤2 and >2 cm), differentiation grade, and microvascular invasion.RESULTS:The mean vascular endothelial growth factor immunoreactivity score was slightly higher in the hepatocellular carcinoma samples (4.83±1.35) than the cirrhotic liver (4.38±1.28) samples. There was a significant and direct correlation between these mean scores (rs=0.645, p=0.0001). Cyclooxygenase-2 was expressed in all the cirrhotic liver samples but was only found in 78% of the hepatocellular carcinoma samples. The mean cyclooxygenase-2 score was higher in the cirrhotic liver samples (4.85±1.38) than the hepatocellular carcinoma samples (2.58±1.68), but there was no correlation between the scores (rs=0.177, p=0.23). The mean CD105 percentage in the hepatocellular carcinoma samples (11.2%) was lower than that in the cirrhotic samples (16.9%). There was an inverse relationship in MVD-CD105 expression between the hepatocellular carcinoma and cirrhotic samples (rs=-0.78, p=0.67). There were no significant associations between vascular endothelial growth factor expression and morphological characteristics. Cyclooxygenase-2 and CD105 were associated with hepatocellular carcinoma differentiation grade (p=0.003 and p=0.05, respectively).CONCLUSION:Vascular endothelial growth factor, cyclooxygenase-2, and MVD-CD105 were highly expressed in cirrhotic liver compared to hepatocellular carcinoma and might be involved in liver carcinogenesis. Additionally, cyclooxygenase-2 and CD105 might be involved in hepatocellular carcinoma differentiation grade.

The carboxy-terminal domain of connexin43 (CT-Cx43) modulates the expression of p53 by altering miR-125b expression in low-grade human breast cancers.

Connexin 43 (Cx43) is a widely expressed gap junction protein. It can also regulate various gap-junction independent processes, including cellular proliferation. The latter regulatory functions have been attributed to its carboxy-terminal domain, CT-Cx43. CT-Cx43 has been found to be expressed independent of full-length Cx43 in various cell types. Its nuclear localization has additionally raised the possibility that it may regulate the expression of particular genes, including miRNAs, known play a role in the regulation of cellular proliferation. Here, we set out to uncover the molecular mechanism(s) underlying CT-Cx43 mediated gene (de-)regulation in human breast cancer. Western blotting and quantitative real time PCR were carried to assess the expression of CT-Cx43 and miR-125b in a panel of 60 primary human breast cancer tissues and its paired normal adjacent tissues. In addition, CT-Cx43 was exogenously expressed in the breast cancer-derived cell line MCF-7 and its effect on the expression of miR-125b and its downstream target p53 were evaluated, as well as its effect on cellular proliferation and death using MTT and LDH assays, respectively. We found that CT-Cx43, but not full-length Cx43, was down-regulated in low grade human breast cancers. In addition, we found that the tumor suppressor protein p53 exhibited a decreased expression in theCT-Cx43 down-regulated samples. Interestingly, we found that miR-125b, a negative regulator of p53, exhibited an inverse expression relationship with CT-Cx43 in the breast cancer samples tested. This inverse relationship was confirmed by exogenous expression of CT-Cx43 in MCF-7 cells. In addition, we found that CT-Cx43 up-regulation and subsequent miR-125b down-regulation resulted in a decreased proliferation of MCF-7 cells. Our data suggest a mechanism by which CT-Cx43 may regulate cell proliferation. Targeting of CT-Cx43 and/or miR-125b may be instrumental for therapeutic intervention in human breast cancer.

Connecting rules from paired miRNA and mRNA expression data sets of HCV patients to detect both inverse and positive regulatory relationships.

BackgroundIntensive research based on the inverse expression relationship has been undertaken to discover the miRNA-mRNA regulatory modules involved in the infection of Hepatitis C virus (HCV), the leading cause of chronic liver diseases. However, biological studies in other fields have found that inverse expression relationship is not the only regulatory relationship between miRNAs and their targets, and some miRNAs can positively regulate a mRNA by binding at the 5' UTR of the mRNA.ResultsThis work focuses on the detection of both inverse and positive regulatory relationships from a paired miRNA and mRNA expression data set of HCV patients through a 'change-to-change' method which can derive connected discriminatory rules. Our study uncovered many novel miRNA-mRNA regulatory modules. In particular, it was revealed that GFRA2 is positively regulated by miR-557, miR-765 and miR-17-3p that probably bind at different locations of the 5' UTR of this mRNA. The expression relationship between GFRA2 and any of these three miRNAs has not been studied before, although separate research for this gene and these miRNAs have all drawn conclusions linked to hepatocellular carcinoma. This suggests that the binding of mRNA GFRA2 with miR-557, miR-765, or miR-17-3p, or their combinations, is worthy of further investigation by experimentation. We also report another mRNA QKI which has a strong inverse expression relationship with miR-129 and miR-493-3p which may bind at the 3' UTR of QKI with a perfect sequence match. Furthermore, the interaction between hsa-miR-129-5p (previous ID: hsa-miR-129) and QKI is supported with CLIP-Seq data from starBase. Our method can be easily extended for the expression data analysis of other diseases.ConclusionOur rule discovery method is useful for integrating binding information and expression profile for identifying HCV miRNA-mRNA regulatory modules and can be applied to the study of the expression profiles of other complex human diseases.

Involvement of hepatocyte growth factor-induced epithelial-mesenchymal transition in human adenomyosis.

Adenomyosis is commonly believed to arise from the basalis endometrium. As an estromedin growth factor, hepatocyte growth factor (HGF) exhibits multiple functions in endometriosis, a disease commonly believed to arise from the functionalis endometrium. Here, we investigated the role of HGF in the occurrence of epithelial-mesenchymal transition (EMT) in adenomyosis. Full-thickness-biopsy specimens from endometrium to myometrium were collected after hysterectomy from women with and without adenomyosis. The relationship between HGF and E-cadherin (epithelial cell marker) and N-cadherin (mesenchymal cell markers) was examined at the gene and protein levels using endometrial epithelial cells (EECs) in culture and tissues by quantitative RT-PCR and immunohistochemistry. The gene and protein expressions of two transcriptional repressors of E-cadherin, SLUG and SNAIL, were examined using Ishikawa cells and in response to HGF and estrogen (E2). HGF down-regulated E-cadherin and up-regulated N-cadherin mRNA expression in EECs, and an inverse relationship in protein expression between HGF and E-cadherin was observed in basalis endometria derived from women with diffuse and focal adenomyosis. HGF induced morphological changes of EECs from a cobblestone-like appearance to spindle-shaped cells and promoted migration of EECs. Ishikawa cells exhibited up-regulation of SLUG/SNAIL gene expression in response to both HGF and E2 with an additive effect between them. HGF- and E2-promoted SLUG/SNAIL gene expression was significantly abrogated after pretreatment of cells with anti-HGF antibody or ICI 182720, an estrogen receptor antagonist. HGF may be involved in gland invagination deep into the myometrium by inducing EMT at the endo-myometrial junction in women with adenomyosis.

Role of microRNA modulation in the interferon-α/ribavirin suppression of HIV-1 in vivo.

BackgroundInterferon-α (IFN-α) treatment suppresses HIV-1 viremia and reduces the size of the HIV-1 latent reservoir. Therefore, investigation of the molecular and immunologic effects of IFN-α may provide insights that contribute to the development of novel prophylactic, therapeutic and curative strategies for HIV-1 infection. In this study, we hypothesized that microRNAs (miRNAs) contribute to the IFN-α-mediated suppression of HIV-1. To inform the development of novel miRNA-based antiretroviral strategies, we investigated the effects of exogenous IFN-α treatment on global miRNA expression profile, HIV-1 viremia, and potential regulatory networks between miRNAs and cell-intrinsic anti-HIV-1 host factors in vivo.MethodsGlobal miRNA expression was examined in longitudinal PBMC samples obtained from seven HIV/HCV-coinfected, antiretroviral therapy-naïve individuals before, during, and after pegylated interferon-α/ribavirin therapy (IFN-α/RBV). We implemented novel hybrid computational-empirical approaches to characterize regulatory networks between miRNAs and anti-HIV-1 host restriction factors.ResultsmiR-422a was the only miRNA significantly modulated by IFN-α/RBV in vivo (p<0.0001, paired t test; FDR<0.037). Our interactome mapping revealed extensive regulatory involvement of miR-422a in p53-dependent apoptotic and pyroptotic pathways. Based on sequence homology and inverse expression relationships, 29 unique miRNAs may regulate anti-HIV-1 restriction factor expression in vivo.ConclusionsThe specific reduction of miR-422a is associated with exogenous IFN-α treatment, and likely contributes to the IFN-α suppression of HIV-1 through the enhancement of anti-HIV-1 restriction factor expression and regulation of genes involved in programmed cell death. Moreover, our regulatory network analysis presents additional candidate miRNAs that may be targeted to enhance anti-HIV-1 restriction factor expression in vivo.

Deep parallel sequencing reveals conserved and novel miRNAs in gill and hepatopancreas of giant freshwater prawn

MicroRNAs (miRNAs) are ∼20–22 nucleotides, non protein-coding RNA regulatory genes that post-transcriptionally regulate many protein-coding genes, influencing critical biological and metabolic processes. While the number of known microRNA is increasing, there is currently no published data for miRNA from giant freshwater prawns, Macrobrachium rosenbergii (M. rosenbergii), a commercially cultured and economically important food species. In this study, we identified novel miRNAs in the gill and hepatopancreas of M. rosenbergii. Through a deep parallel sequencing analysis and an in silico data analysis approach, 327 miRNA families were identified from small RNA libraries with reference to both the de novo transcriptome of M. rosenbergii obtained from RNA-Seq and to miRBase (Release 18.0, November 2012). Based on the identified mature miRNA and recovered precursor sequences that form appropriate hairpin structures, three conserved miRNA (miR125, miR750, miR993) and 27 novel miRNA candidates encoding messenger-like non-coding RNA were identified. miR-125, miR-750, G-m0002/H-m0009, G-m0005, G-m0008/H-m0016, G-m0011/H-m0027 and G-m0015 were selected for experimental validation with stem-loop quantitative RT-PCR and were found to be coherent with the expression profile of deep sequencing data as evaluated with Pearson's correlation coefficient (r = 0.835178 for miRNA in gill, r = 0.724131 for miRNA in hepatopancreas). Using a combinatorial approach of pathway enrichment analysis and inverse expression relationship of miRNA and mRNA, four co-expressed novel miRNA candidates (G-m0005, G-m0008/H-m0016, G-m0011/H-m0027, and G-m0015) were found to be associated with energy metabolism. In addition, the expression of the three novel miRNA candidates (G-m0005, G-m0008/H-m0016, and G-m0011/H-m0027) were also found to be significantly reduced at 9 and 24 h post infection in M. rosenbergii challenged with infectious hypodermal and hematopoietic necrosis virus, suggesting a functional role of these miRNAs in crustacean immune defense.

X-linked inhibitor of apoptosis protein and active caspase-3 expression patterns in antral follicles in the sheep ovary

X-linked inhibitor of apoptosis protein (XIAP) interacts with caspases to inhibit their activity, thereby providing a potential mechanism for regulation of granulosa cell apoptosis occurring during follicular atresia. The aim of this study was to determine the presence and localization of XIAP mRNA and protein content in the sheep ovary and compare these expression patterns with active caspase-3 protein in the same antral follicles. Romney ewe estrous cycles (n=25) were synchronized with 2-3 Estrumate injections and ovarian tissue collected during the luteal and follicular phases of the cycle. The presence of XIAP mRNA was confirmed by RT-PCR using laser capture microdissected ovarian cell samples. XIAP mRNA was subsequently localized by in situ hybridization histochemistry and XIAP and active caspase-3 protein visualized by immunohistochemistry. In antral follicles extensive XIAP localization was evident in both granulosa and thecal cells. In contrast, mRNA expression was widespread in granulosa cells and only detected in thecal tissue from a small proportion of antral follicles. Active caspase-3 and XIAP comparative expression analysis showed positive XIAP mRNA expression in all late luteal phase (day 14) follicles, despite varying levels of active caspase-3 protein. A proportion of follicular phase (days 15 and 16) follicles, however, showed an inverse expression relationship at the protein and mRNA levels in both granulosa and thecal tissue, as did XIAP protein in day 14 follicles. These results suggest high XIAP may prevent activation of caspase-3, thereby regulating follicular atresia in antral follicles and could potentially be utilized as a marker of follicular health.